Prognostic Signifcance Of Tumor-infltrating Immune Cells And PD-L1 Expression In Esophageal Squamous Cell Carcinoma

Objective: Immune checkpoint blockade has become the most exciting and highprofile breakthrough in cancer treatment by rapidly changing the treatment pattern of malignant tumors.Programmed death-1 receptor(PD-1)/programmed death-ligand 1(PD-L1)blockade exhibited a remarkable curative effect in some patients.Recently,clinical trials focusing on PD-1/PD-L1 inhibitors are fully carried out in china.To explore the prognostic signifcance of PD-L1 expression and tumor-infltrating immune cells(TILs)for ESCC,we investigated the extent of PD-L1 expression and TILs in formalin-fxed samples from ESCC patients,and analyzed the association between PDL1 expression,TIL density and outcome.Methods: Archival formalin-fxed,paraffn-embedded ESCC samples were collected from patients diagnosed with primary ESCC after surgery or by endoscopic biopsy at the Affliated 307 Hospital Cancer Center,Beijing,P.R.China between 2007 and 2015.Serial sections of 4 ?m thickness were obtained from each specimen.Expression of PD-L1 in ESCC tumor specimens was assessed by IHC with an Autostainer Plus(DakoAgilent Technologies),utilizing a proprietary PD-L1 antibody(rabbit monoclonal antibody clone 73-10,Merck KGaA,Darmstadt,Germany)at 1:1000 dilution.Frequency of PD-L1+ cells and staining intensity were analyzed.In tumor cells,PD-L1+ was defined by three cut off,1%,5% and 25%.The degree of infltration by TILs was evaluated from the H&E-stained specimens and scored from 0 to 3: 0 = none;1 = rare;2 = moderate and focal infltration;and 3 = prominent and diffuse infiltration(high).Histopathologic data was obtained from pathology reports,and patient survival data was obtained from the patient's attending physician.Results: A total of 428 ESCC specimens,one from each patient,were obtained from patients admitted to the Affliated 307 Hospital Cancer Center,Beijing,P.R.China during 2007 to 2015.Samples were collected either by surgery(30.6%,n=131)or by endoscopic biopsy(69.4%,n=297)from treatment-na?ve patients.PD-L1 expression in ESCC specimens.Among 428 ESCC specimens,79.7%,32.7% and 9.1% were PD-L1+ tumors using cutoff values of 1%,5% and 25%,respectively.There was no signifcant difference in percentages of PD-L1+ specimens tumors between those obtained via surgery or biopsy(102/131,77.9% PDL1+ in surgical specimens versus 239/297,80.5% PD-L1+ in biopsy specimens,p=0.602).Statistical analyses using Spearman Rank Correlation indicated that PD-L1+ specimens were associated with aggressive clinicopathological features,which included deeper tumor invasion(p=0.037)and nodal metastasis(p=0.013).DFS and OS was signifcantly related to PD-L1 expression(p=0.001;p=0.039),PD-L1+ patients exhibited worse DFS and OS than PD-L1– patients,median DFS and median OS was 37.5 months vs.13.8 months,44.5 months vs.24.2months,respectively.Patients in the palliative treatment cohort exhibited no signifcant association between PD-L1 expression and PFS(p=0.990)or OS(p=0.736).TIL density and patient prognosis.TIL density in 428 ESCC specimens were 6.3%(TILs=0,n=27),30.6%(TILs=1,n=131),46.3%(TILs=2,n=198),18.8%(TILs=3,n=72),respectively.Similar to PD-L1 expression,there was no signifcant difference in TIL density between surgical and biopsy specimens(p=0.243).There was no signifcant association between DFS or OS in the four different TILs density cohort.However,patients who received esophagectomy who had a high TIL score(3)showed a signifcantly better OS than those with a moderate(2),low(1)or none(0)TIL score(HR=2.264,95% CI: 1.039,4.936,p=0.039).Classifcation based on TILs and PD-L1 expression.Based on PD-L1 status and TIL density,ESCC specimens were classifed into 4 groups.Type I was PD-L1+ and TILs+(PD-L1+ with TILs driving adaptive immune resistance),type II was PDL1– and TILs–(PD-L1–with no TILs indicating immune ignorance),type III was PD-L1+ and TILs–(PD-L1+ with no TILs indicating intrinsic induction),and type IV was PD-L1– and TILs+(PD-L1– with TILs indicating the role of other suppressor(s)in promoting immune tolerance).The proportions of four types were 13.6%(58/428),17.1%(73/428),66.1%(283/428),and 3.3%(14/428),respectively.There was a significant difference in OS among four types,with median OS of 30.8,24.1,18.3 and 27.0 months respectively for the four types(p=0.00).OS was longer for type I,II and IV than type III(p=0.040,p=0.034,p=0.005).Statistical analyses of independent factors associated with DFS and OS in defnitive treatment cohort indicated that DFS was signifcantly related to TNM status(HR=1.541,95% CI: 1.034,2.229,p=0.034)and PD-L1 expression(HR=2.336,95% CI: 1.441,3.106,p=0.001).Similarly,OS was negatively correlated with PD-L1 expression(HR=1.873,95% CI: 1.155,2.331,p=0.01)and TNM status(HR=1.413,95% CI: 0.879,2.602,p=0.040).Independent factors associated with OS palliative treatment cohort were age(HR=0772,95%CI:0.585,1.020;p=0.019),TNM stage(HR=1.066,95%CI:0.782,1.453;p=0.002),chemotherapy regimen(HR=1.764,95%CI:1.091,2.854;p=0.021)and chemotherapy cycles(HR=1.877,95%CI:1.252,2.812;p=0.002).Conclusion: PD-L1 expression in ESCC specimens could be detected by IHC with a high positive percentage.Analysis of the correlation between PDL1 expression and clinicopathologic features suggests that tumor PD-L1 positivity might be associated with more aggressive tumor progression,including deeper tumor invasion and more nodal metastasis.Patients with PD-L1+ tumors were more likely to have worse outcomes,including those who underwent surgery or(chemo-)radiation.High TILs were associated with a longer OS in patients who underwent surgery.Type I(PDL1+/TIL+)and type III(PD-L1+/TIL-)were believed to be largely responding to checkpoint blockade.